The active role of the endothelium in acute inflammatory processes has only recently been appreciated. In particular, the identification and molecular cloning of the cytokine regulated endothelial-leukocyte adhesion molecules, ELAM-1 and ICAM-1, provides a basis to understand the cellular mechanisms involved in leukocyte-endothelial interactions during inflammation. Preliminary studies by the applicant have demonstrated that cytokine- activated endothelial monolayers promote an upregulated neutrophil transmigration and that expression of both ELAM-1 and ICAM-1 are required for this process. This project will examine the cellular and molecular mechanisms of neutrophil activation during their adhesive interactions with cytokine activated endothelium, focusing on the role of ELAM-1. The primary goals of this project are to: determine whether neutrophils become activated during adhesive interactions with cytokine-activated endothelial monolayers as measured by biochemical (e.g., mobilization of cytosolic Ca2+ by microspectrofluorimetry) and morphological (time lapse videomicroscopy) parameters; 2) to determine whether purified ELAM-1 (human recombinant ELAM-1) deposited on inert surfaces can directly activate neutrophils using the same indices as above; 3) to determine which epitopes on ELAM-1 are involved in neutrophil activation using monoclonal antibodies which recognize various epitopes on ELAM-1; 4) using an in vitro transmigration assay, assess the role of ELAM-1-dependent neutrophil activation in neutrophil activation. Thus, the proposed studies will combine cell biological techniques and immunological approaches to gain insight into endothelial-dependent mechanisms important in the local regulation of inflammation, i.e., neutrophil adhesion and transmigration.